Methods, Compositions, and Kits for Spatial Detection of Target Nucleic Acids

This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/655,740 filed on Jun. 4, 2024, the contents of which are hereby incorporated by reference.

This application contains a Sequence Listing that has been submitted electronically as an XML file named “47706-0392001_SL_ST26.XML.” The XML file, created on May 30, 2025, is 32,243 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

Cells within a tissue of a subject have differences in cell morphology and/or function due to varied analyte levels (e.g., gene and/or protein expression) within the different cells. The specific position of a cell within a tissue (e.g., the cell's position relative to neighboring cells or the cell's position relative to the tissue microenvironment) can affect, e.g., the cell's morphology, differentiation, fate, viability, proliferation, behavior, signaling and cross-talk with other cells in the tissue.

Spatial heterogeneity has been previously studied using techniques that only provide data for a small handful of analytes in the context of an intact tissue or a portion of a tissue, or provide substantial analyte data for dissociated tissue (i.e., single cells), but fail to provide information regarding the position of the single cell in a parent biological sample (e.g., tissue sample).

Various types of tissues often require different treatment conditions, e.g., permeabilization conditions such as the use of different enzymes, duration, and additional reagents (e.g., detergents, surfactants), etc., when performing spatial analyses based on factors such as extracellular matrix proteins among others. Resources such as reagents, time, and sequencing costs can be wasted when determining optimal conditions for spatial analysis.

Improved methods and conditions are still needed, which can be applied across various tissue types without excessive experimental testing.

The present disclosure features methods, compositions, and kits to determine the location of target nucleic acids in a biological sample. The methods described herein can be used across various species and/or types of tissue, thereby foregoing the need for tissue specific permeabilization optimization. For example, human breast cancer is typically rich in collagenase and can require additional permeabilization condition testing to determine optimal conditions to perform spatial transcriptomic analyses. Thus, the disclosed methods, kits and compositions can be useful in performing spatial analysis without the need for tissue permeabilization optimization. The present disclosure utilizes, in some embodiments, an in situ reverse transcription, followed by tagmentation, and capture of the fragmented products on a spatial array as further described herein.

Thus provided herein are methods for determining a location of a target RNA in a biological sample, the method including: a) hybridizing a primer to the target RNA in the biological sample; b) extending the primer using the target RNA as a template to provide a cDNA hybridized to the target RNA, thereby generating a cDNA:RNA duplex; c) incorporating at least three untemplated nucleotides at a 3′ end of the cDNA of the cDNA:RNA duplex; d) hybridizing a first adapter to the at least three untemplated nucleotides and extending the cDNA of the cDNA:RNA duplex using the first adapter as a template, thereby generating an extended cDNA:RNA duplex; e) contacting a transposome complex with the biological sample to insert a second adapter into the extended cDNA:RNA duplex, thereby generating a 5′ fragmented cDNA:RNA duplex; f) releasing the RNA from the 5′ fragmented cDNA:RNA duplex, thereby generating a 5′ cDNA molecule including (i) a complement of the first adapter, and (ii) the second adapter; g) hybridizing the first adapter of the 5′ cDNA molecule to a capture domain of a capture probe in an array including a plurality of capture probes, where the capture probe includes: (i) a spatial barcode and (ii) a capture domain; and h) determining the sequence of (i) the spatial barcode, or a complement thereof, (ii) the 5′ cDNA molecule or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the target RNA in the biological sample.

In some embodiments, the biological sample is disposed on the array or where the biological sample is disposed on a first substrate. In some embodiments, when the biological sample is disposed on a first substrate, the method includes aligning the first substrate including the biological sample with a second substrate including the array, such that at least a portion of the biological sample is aligned with at least a portion of the array, and migrating the 5′ cDNA molecule from the biological sample to the array, optionally where the migrating includes electrophoresis.

In some embodiments, the extending in step (b) includes use of a reverse transcriptase, where incorporating the at least three untemplated nucleotides includes use of the reverse transcriptase. In some embodiments, incorporating the at least three untemplated nucleotides includes use of a terminal transferase, optionally where the terminal transferase is a terminal deoxynucleotidyl transferase.

In some embodiments, the first adapter includes RNA.

In some embodiments, the at least three untemplated nucleotides include a homopolynucleotide sequence or a heteropolynucleotide sequence.

In some embodiments, the second adapter sequences include a functional domain, optionally where the functional domain includes a primer binding site.

In some embodiments, the transposome complex includes a transposase enzyme, a transposon sequence, and the second adapter; and optionally where the transposase enzyme is a Tn5 transposase enzyme, a Mu transposase enzyme, a Tn7 transposase enzyme, a Vibrio species transposase, or functional derivatives thereof.

In some embodiments, releasing the RNA includes use of heat, potassium hydroxide, or an RNase, optionally where the RNase include one or more of RNase A, RNase C, RNase H, and RNase I.

In some embodiments, the second adapter is inserted at a 5′ end of the cDNA in the 5′ fragmented cDNA:RNA duplex.

In some embodiments, step (e) includes generating a 3′ fragmented cDNA:RNA duplex and one or more middle fragmented cDNA:RNA duplexes, the method including a reverse transcription reaction to gap-fill the 3′ fragmented cDNA:RNA duplex and the one or more middle fragmented cDNA:RNA duplexes.

In some embodiments, releasing the RNA from the 3′ fragmented cDNA:RNA duplex and the one or more middle fragmented cDNA:RNA duplexes, thereby generating a 3′ cDNA molecule and one or more middle cDNA molecule(s), respectively.

In some embodiments, the array includes a second plurality of capture probes, where a second capture probe of the second plurality of capture probes includes: (i) a second spatial barcode and (ii) a second capture domain; where the second adapter includes a sequence complementary to the second capture domain.

In some embodiments, the method includes hybridizing the second adapter of the 3′ cDNA molecule to the second capture domain, and determining the sequence of: (i) the second spatial barcode, or a complement thereof, and (ii) the 3′ cDNA molecule, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the target RNA in the biological sample.

In some embodiments, the method includes extending the capture probe using the 5′ cDNA molecule as a template and/or extending the 5′ cDNA molecule using the capture probe as a template.

In some embodiments, the determining step includes sequencing.

In some embodiments, the method includes permeabilizing, staining, and/or imaging the biological sample, and where the biological sample is a tissue section, optionally a fresh-frozen tissue section or a fixed tissue section.

In some embodiments, the target RNA is mRNA.

Also provided herein are methods for processing a target RNA in a biological sample, the method including: a) hybridizing a primer to the target RNA in the biological sample; b) extending the primer using the target RNA as a template to provide a cDNA hybridized to the target RNA, thereby generating a cDNA:RNA duplex; c) incorporating at least three untemplated nucleotides at a 3′ end of the cDNA of the cDNA:RNA duplex; d) hybridizing a first adapter to the at least three untemplated nucleotides and extending the cDNA of the cDNA:RNA duplex using the first adapter as a template, thereby generating an extended cDNA:RNA duplex; e) contacting a transposome complex with the biological sample to insert a second adapter into the extended cDNA:RNA duplex, thereby generating a 5′ fragmented cDNA:RNA duplex; and f) releasing the RNA from the 5′ fragmented cDNA:RNA duplex, thereby generating a 5′ cDNA molecule including a complement of the first adapter and the second adapter.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

The term “about” or “approximately” as used herein means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably still up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value.

The term “substantially complementary” used herein means that a first sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-40, 40-60, 60-100, or more nucleotides, or that the two sequences hybridize under stringent hybridization conditions. Substantially complementary also means that a sequence in one strand is not completely and/or perfectly complementary to a sequence in an opposing strand, but that sufficient bonding occurs between bases on the two strands to form a stable hybrid complex in set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using the sequences and standard mathematical calculations known to those skilled in the art.

The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise. Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. Like reference symbols in the drawings indicate like elements.

shows an exemplary sandwiching process where a first substrate (e.g., a slide), including a biological sample, and a second substrate (e.g., array slide) are brought into proximity with one another.

shows a fully formed sandwich configuration creating a chamber formed from the one or more spacers, the first substrate, and the second substrate.

shows a perspective view of an exemplary sample handling apparatus in a closed position.

shows a perspective view of an exemplary sample handling apparatus in an open position.

shows the first substrate angled over (superior to) the second substrate.

shows that as the first substrate lowers, and/or as the second substrate rises, the dropped side of the first substrate may contact a drop of reagent medium.

shows a full closure of the sandwich between the first substrate and the second substrate with one or more spacers contacting both the first substrate and the second substrate.

shows a side view of the angled closure workflow.

shows a top view of the angled closure workflow.

is a schematic diagram showing an example of a barcoded capture probe, as described herein.

shows a schematic illustrating a cleavable capture probe.

shows exemplary capture domains on capture probes.

shows an exemplary arrangement of barcoded features within an array.

shows and exemplary workflow for performing templated capture and producing a ligation product, andshows an exemplary workflow for capturing a ligation product fromon a substrate.

is a schematic diagram of an exemplary analyte capture agent.

is a schematic diagram depicting an exemplary interaction between a feature-immobilized capture probeand an analyte capture agent.

from top to bottom is a schematic showing an example of an in situ reverse transcribed cDNA product hybridized to a target RNA (top) followed by three different cDNA:RNA fragments (e.g., a 5′ fragment, a 3′ fragment, and a middle fragment) generated after tagmentation.

is a schematic showing remnants of the three different cDNA:RNA fragments (e.g., a 5′ fragment, a 3′ fragment, and a middle fragment) fromafter releasing the target RNA from the cDNA:RNA fragments.(bottom) shows capture of the 5′ fragment via a capture probe including a capture domain on a substrate.